Glioblastoma multiforme(GBM) is a devastating disease that almost invariably leads to patient death despite best efforts using standard therapies that include surgery, radiation and chemotherapy. New therapeutic interventionsare needed which may be used in combination with standard medical practice. Among these treatments, gene therapy potentially holds promise for treatment of GBM however impediments to effective gene delivery remain. Highly attenuated replication competent HSV-1 vectors providea powerful opportunityto provide effective gene delivery in additionto the natural lytic features (oncolysis) and early phase human trials support the safety of this approach. In this proposal, experiments are outlinedto explore methodsto enhancethe potency of HSV oncolyticvectors through improvedvector distribution within the tumor mass, the testing of additional mutant vector backbones whose performance may be improved by enhanced and more specific intra-tumoralreplicationand through the use of additionaltransgenes that may be more effective in destruction of the tumor mass including locally infiltrating tumor cells into normal brain tissue. Throughout this investigationvector performanceand tumor killing will be evaluated in combinationwith radiosurgery. We will use the performance of G207 as a benchmark with which to compare any vector improvements. In four related specific aims we will: (i) Exploit the use of collagenases to enhance intra-tumoral vector distribution as visualized by advanced vital microscopy; (ii) Examine new genetic alterations in the HSV genome in a search for more active mutant oncolytic vectors that have the same or better safety profiles as vectors currently used in early phasepatient studies (e.g. G207); (iii) Develop retargeting strategies to enable tumor-specific HSV infection through recognition of tumor-cell receptors; and (iv) Introduce novel anti-tumor transgenes into the vector backbone that include (a) purine nucleoside phosphorylase (PNP) in combination with 6-methylpurine(MeP) treatment, (b) chlorotoxin (CltX), a peptide that inhibits tumor cell migration and may induce tumor cell apoptosis (c) tumor necrosis factor (TNFoc)that acts to sensitize tumor cells and the tumor vasculature to radiosurgical methods. The outcome of these studies are intended to discover new vectors, more effective transgenes and delivery strategies which together may provide gene therapy as an effective approach to at least prolong the survival of patients with recurrent GBM over currently available treatment methods.